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The role of autophagy as a mechanism of toxicity induced by multi-walled carbon nanotubes in human lung cells.

Tsukahara T, Matsuda Y, Haniu H - Int J Mol Sci (2014)

Bottom Line: Although autophagy is considered as a cytoprotective process, it is often observed in association with cell death, and the relationship between autophagy and cell death remains unclear.Our recent study suggests that the levels of autophagy-related genes (LC3B) and autophagosome formation are clearly up-regulated, along with an increase in numbers of autophagosome vacuoles.This review highlights the importance of autophagy as an emerging mechanism of CNT toxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. ttamotsu@nagasaki-u.ac.jp.

ABSTRACT
Carbon nanotubes (CNTs) are promising nanomaterials having unique physical and chemical properties, with applications in a variety of fields. In this review, we briefly summarize the intrinsic properties of highly purified multi-walled CNTs (MWCNTs, HTT2800) and their potential hazardous effects on intracellular and extracellular pathways, which alter cellular signaling and impact major cell functions such as differentiation, reactive oxygen species (ROS) production, apoptosis, and autophagy. A recent study suggested that the induction of autophagy by CNTs causes nanotoxicity. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with exposure to CNTs. Although autophagy is considered as a cytoprotective process, it is often observed in association with cell death, and the relationship between autophagy and cell death remains unclear. Our recent study suggests that the levels of autophagy-related genes (LC3B) and autophagosome formation are clearly up-regulated, along with an increase in numbers of autophagosome vacuoles. This review highlights the importance of autophagy as an emerging mechanism of CNT toxicity.

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Related in: MedlinePlus

Carbon nanotubes (CNT)-induced autophagy. Schematic representation of the proposed mechanism for HTT2800. During autophagy, a double-layered, membrane-bound autophagosome is formed that surrounds the proteins and damaged organelles identified for degradation. When autophagy is blocked by 3-MA, caspase-3 activation is induced. Autophagy inhibition accelerates apoptosis in cells. Furthermore, HTT2800-induced autophagic cell death occurred in the absence of caspase activation, via an ROS-independent pathway. These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells.
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ijms-16-00040-f001: Carbon nanotubes (CNT)-induced autophagy. Schematic representation of the proposed mechanism for HTT2800. During autophagy, a double-layered, membrane-bound autophagosome is formed that surrounds the proteins and damaged organelles identified for degradation. When autophagy is blocked by 3-MA, caspase-3 activation is induced. Autophagy inhibition accelerates apoptosis in cells. Furthermore, HTT2800-induced autophagic cell death occurred in the absence of caspase activation, via an ROS-independent pathway. These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells.

Mentions: Autophagosome accumulation induced by CNT treatment was found to be associated with cell death in a majority of published reports [40]. However, the possibility of autophagy inhibition has not been adequately investigated. We recently reported that HTT2800 has emerged as a novel class of autophagy inducers [18]. Autophagy is a tightly regulated cellular process involving bulk cytoplasmic and organelle degradation [41,42,43]. Common to nearly all eukaryotes, autophagy is a lysosomal degradation pathway that recycles intracellular components such as protein aggregates and damaged or dysfunctional intracellular organelles [41]. CNTs have been detected in lysosomes upon internalization, and they have been found to be associated with lysosomal dysfunction [33]. Autophagy was recently recognized as a critical cell death pathway and autophagosome accumulation was found to be associated with exposure to various nanoparticles [17,44]. In eukaryotes, autophagy is the main catabolic mechanism by which the cell degrades cytoplasmic components that are engulfed in double membrane-bound vesicles known as autophagosomes [43]. In the first step of autophagosome development, the cytoplasmic particulate matter is sequestered and then the double-membrane autophagosome fuses with a lysosome to form the autolysosome. To date, three forms of autophagy have been identified: (1) chaperone-mediated autophagy; (2) microautophagy; and (3) macroautophagy, which differ in the mode of delivery to the lysosome [45]. Our recent study suggested that HTT2800-induced autophagy in BEAS-2B cells was based on expression of the autophagic marker, light-chain 3 (LC3) and its effect on cell death and proliferation [18]. This characteristic conversion of LC3 can be used to monitor autophagic activity. LC3 was originally identified as a subunit of microtubule-associated proteins 1A and 1B [46], which associated with the autophagosome membranes after processing. Cleavage of LC3 at the carboxyl terminus occurs immediately following synthesis of the cytosolic form of non-lipidated LC3B-I (19 kDa). During autophagy, LC3-I is converted to lipidated LC3B-II (16 kDa), which is tightly bound to the membrane [46]. HTT2800 significantly induced LC3B-II expression, which decreased after treatment with an autophagy inhibitor, 3-methyladenine (3-MA) [18]. Furthermore, we monitored the cellular distribution of green fluorescent protein-tagged LC3B using fluorescence microscopy. We found that in contrast to the cytoplasmic localization of LC-3B-I, LC3-BII associated with both the outer and inner membranes of the autophagosome and the number of vacuoles was significantly lower in untreated cells than in HTT2800-treated human lung cells [18]. We further examined whether endocytosis and lysosomal regulation might be involved in HTT2800-induced cell death. Prior to HTT2800 exposure, we treated cells with brefeldin A, which inhibits the transport of proteins from the endoplasmic reticulum to the Golgi; 3-MA, which inhibits autophagosome formation; or E64-d and pepstatin A (E64-d + pepstatin A), which are lysosomal proteinase inhibitors. Interestingly, 3-MA and E64-d + pepstatin A, but not brefeldin A, provided protection against the HTT2800-induced cell death that occurred via the autophagic rather than the endocytic pathway [18]. In addition, pretreatment with the caspase inhibitor, z-VAD-FMK, had no significant protective effect on HTT2800-induced inhibition of cell growth. Interestingly, we found that caspase-3 activation was induced in 3-MA-treated BEAS-2B cells [18]. It is also possible that CNTs induce autophagy via an oxidative stress and inflammatory response, such as accumulation of damaged proteins due to a decrease in autophagy flux. It has been reported that the disruption of autophagy flux could lead to enhanced NLRP3 inflammasome accumulation and exaggerated IL-1b production leading to lung fibrosis [47]. These results suggested that autophagy modulation mediated by CNTs may lead to autophagy-mediated cell death and could serve as a specific signal for lung diseases. In conclusion, these studies taken together, suggest that autophagy plays a functional role in the stress response to CNT exposure in vitro, and that CNTs might also contribute to autophagic cell death (Figure 1). These markers could serve as possible therapeutic targets that could be explored further in the clinical management of lung diseases.


The role of autophagy as a mechanism of toxicity induced by multi-walled carbon nanotubes in human lung cells.

Tsukahara T, Matsuda Y, Haniu H - Int J Mol Sci (2014)

Carbon nanotubes (CNT)-induced autophagy. Schematic representation of the proposed mechanism for HTT2800. During autophagy, a double-layered, membrane-bound autophagosome is formed that surrounds the proteins and damaged organelles identified for degradation. When autophagy is blocked by 3-MA, caspase-3 activation is induced. Autophagy inhibition accelerates apoptosis in cells. Furthermore, HTT2800-induced autophagic cell death occurred in the absence of caspase activation, via an ROS-independent pathway. These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307234&req=5

ijms-16-00040-f001: Carbon nanotubes (CNT)-induced autophagy. Schematic representation of the proposed mechanism for HTT2800. During autophagy, a double-layered, membrane-bound autophagosome is formed that surrounds the proteins and damaged organelles identified for degradation. When autophagy is blocked by 3-MA, caspase-3 activation is induced. Autophagy inhibition accelerates apoptosis in cells. Furthermore, HTT2800-induced autophagic cell death occurred in the absence of caspase activation, via an ROS-independent pathway. These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells.
Mentions: Autophagosome accumulation induced by CNT treatment was found to be associated with cell death in a majority of published reports [40]. However, the possibility of autophagy inhibition has not been adequately investigated. We recently reported that HTT2800 has emerged as a novel class of autophagy inducers [18]. Autophagy is a tightly regulated cellular process involving bulk cytoplasmic and organelle degradation [41,42,43]. Common to nearly all eukaryotes, autophagy is a lysosomal degradation pathway that recycles intracellular components such as protein aggregates and damaged or dysfunctional intracellular organelles [41]. CNTs have been detected in lysosomes upon internalization, and they have been found to be associated with lysosomal dysfunction [33]. Autophagy was recently recognized as a critical cell death pathway and autophagosome accumulation was found to be associated with exposure to various nanoparticles [17,44]. In eukaryotes, autophagy is the main catabolic mechanism by which the cell degrades cytoplasmic components that are engulfed in double membrane-bound vesicles known as autophagosomes [43]. In the first step of autophagosome development, the cytoplasmic particulate matter is sequestered and then the double-membrane autophagosome fuses with a lysosome to form the autolysosome. To date, three forms of autophagy have been identified: (1) chaperone-mediated autophagy; (2) microautophagy; and (3) macroautophagy, which differ in the mode of delivery to the lysosome [45]. Our recent study suggested that HTT2800-induced autophagy in BEAS-2B cells was based on expression of the autophagic marker, light-chain 3 (LC3) and its effect on cell death and proliferation [18]. This characteristic conversion of LC3 can be used to monitor autophagic activity. LC3 was originally identified as a subunit of microtubule-associated proteins 1A and 1B [46], which associated with the autophagosome membranes after processing. Cleavage of LC3 at the carboxyl terminus occurs immediately following synthesis of the cytosolic form of non-lipidated LC3B-I (19 kDa). During autophagy, LC3-I is converted to lipidated LC3B-II (16 kDa), which is tightly bound to the membrane [46]. HTT2800 significantly induced LC3B-II expression, which decreased after treatment with an autophagy inhibitor, 3-methyladenine (3-MA) [18]. Furthermore, we monitored the cellular distribution of green fluorescent protein-tagged LC3B using fluorescence microscopy. We found that in contrast to the cytoplasmic localization of LC-3B-I, LC3-BII associated with both the outer and inner membranes of the autophagosome and the number of vacuoles was significantly lower in untreated cells than in HTT2800-treated human lung cells [18]. We further examined whether endocytosis and lysosomal regulation might be involved in HTT2800-induced cell death. Prior to HTT2800 exposure, we treated cells with brefeldin A, which inhibits the transport of proteins from the endoplasmic reticulum to the Golgi; 3-MA, which inhibits autophagosome formation; or E64-d and pepstatin A (E64-d + pepstatin A), which are lysosomal proteinase inhibitors. Interestingly, 3-MA and E64-d + pepstatin A, but not brefeldin A, provided protection against the HTT2800-induced cell death that occurred via the autophagic rather than the endocytic pathway [18]. In addition, pretreatment with the caspase inhibitor, z-VAD-FMK, had no significant protective effect on HTT2800-induced inhibition of cell growth. Interestingly, we found that caspase-3 activation was induced in 3-MA-treated BEAS-2B cells [18]. It is also possible that CNTs induce autophagy via an oxidative stress and inflammatory response, such as accumulation of damaged proteins due to a decrease in autophagy flux. It has been reported that the disruption of autophagy flux could lead to enhanced NLRP3 inflammasome accumulation and exaggerated IL-1b production leading to lung fibrosis [47]. These results suggested that autophagy modulation mediated by CNTs may lead to autophagy-mediated cell death and could serve as a specific signal for lung diseases. In conclusion, these studies taken together, suggest that autophagy plays a functional role in the stress response to CNT exposure in vitro, and that CNTs might also contribute to autophagic cell death (Figure 1). These markers could serve as possible therapeutic targets that could be explored further in the clinical management of lung diseases.

Bottom Line: Although autophagy is considered as a cytoprotective process, it is often observed in association with cell death, and the relationship between autophagy and cell death remains unclear.Our recent study suggests that the levels of autophagy-related genes (LC3B) and autophagosome formation are clearly up-regulated, along with an increase in numbers of autophagosome vacuoles.This review highlights the importance of autophagy as an emerging mechanism of CNT toxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. ttamotsu@nagasaki-u.ac.jp.

ABSTRACT
Carbon nanotubes (CNTs) are promising nanomaterials having unique physical and chemical properties, with applications in a variety of fields. In this review, we briefly summarize the intrinsic properties of highly purified multi-walled CNTs (MWCNTs, HTT2800) and their potential hazardous effects on intracellular and extracellular pathways, which alter cellular signaling and impact major cell functions such as differentiation, reactive oxygen species (ROS) production, apoptosis, and autophagy. A recent study suggested that the induction of autophagy by CNTs causes nanotoxicity. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with exposure to CNTs. Although autophagy is considered as a cytoprotective process, it is often observed in association with cell death, and the relationship between autophagy and cell death remains unclear. Our recent study suggests that the levels of autophagy-related genes (LC3B) and autophagosome formation are clearly up-regulated, along with an increase in numbers of autophagosome vacuoles. This review highlights the importance of autophagy as an emerging mechanism of CNT toxicity.

Show MeSH
Related in: MedlinePlus